Value changes of heart function in transesophageal echocardiography were recorded at various time points  after surgery in each group. Myocardial sections were stained with hematoxylin and eosin (H&E) and examined with optical microscope. The degree of myocardial fibrosis was assessed by Sirius Red staining. Terminal dUTP nick end-labeling (TUNEL) and qRT-PCR methods were used to measure the apoptosis rate and to determine the miR-320 expression levels in myocardial tissues. Transesophageal echocardiography showed that the values of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular systolic pressure (LVSP) and ±dp/dtmax in the I/R group were obviously lower than those in the sham group, while the left ventricular end-diastolic pressure (LVEDP) value was higher than that in the sham group. The values of LVEF, LVFS, LVSP and ±dp/dtmax showed a gradual decrease in the I/R group, while the LVEDP value showed an up tendency along with the extension of reperfusion time. The H&E staining revealed that rat myocardial tissue in the I/R group presented extensive myocardial damage; for the I/R + antagomir-320 group, however, the degree of damage in myocardial cells was obviously better than that of the I/R group. The Sirius Red staining results showed that the degree of myocardial fibrosis in the I/R group was more severe along with the extension of the time of reperfusion. For the I/R + antagomir-320 group, the degree of myocardial fibrosis was less severe than that in the I/R group. Tissues samples in both the sham and I/R + antagomir-320 groups showed a lower apoptosis rate compared to I/R group. The qRT-PCR results indicated that miR-320 expression in the I/R group was significantly higher than that in both the sham and I/R + antagomir-320 groups. The expression level of miR-320 is significantly up-regulated in the rat model of myocardial I/R injury, and it may be implicated in the prevention of myocardial I/R injury-triggered left ventricular remodeling.The primary objective of this study investigated the role of microRNA-320 (miR-320) on left ventricular remodeling in the rat model of myocardial ischemia-reperfusion (I/R) injury, and we intended to explore the myocardial mechanism of miR-320-mediated myocardium protection. We collected 120 male Wistar rats (240–280 g) in this study and then randomly divided them into three groups: (1) sham surgery group (sham group:  Cardiovascular disease is the leading cause of death in both the United States and among worldwide populations, according to the World Health Organization . IschemiNumerous evidence suggested that miRs showed evidence of significant roles in regulating gene expression, which are implicated in the cardiac development and pathological process of cardiovascular diseases ,12,13. Tp/dtmax in each group at different time points. The result has shown no significant differences of LVEF, LVFS, LVSP, LVEDP and ±dp/dtmax at different time points in the sham group. Compared with the shame group, the values of LVEF, LVFS, LVSP and ±dp/dtmax in the I/R group at different time points were obviously lower, while the LVEDP value was higher . Furthermore, along with the extension of reperfusion time, the values of LVEF, LVFS, LVSP and ±dp/dtmax in the I/R group gradually declined, but the LVEDP value gradually increased. Nevertheless, the values of LVEF, LVFS, LVSP and ±dp/dtmax of the I/R + antagomir-320 group were higher than those of the I/R group at each time point, but the value of LVEDP was lower than that of the I/R group at each time point   .The H&E staining revealed that the rat myocardial tissue of the sham group had no obvious myocardial injury. Normal myocardial cell and nuclei, mild interstitial congestion and a few wavy fibers were observed in the sham group without significant differences at different time points. Compared with the sham group, the I/R group exhibited extensive myocardial damage—disruption and lysis of the myocardial cell, proliferation of fiber cells, formation of an infarct scar region—while the degree of damage in myocardial cells of the I/R + antagomir-320 group was significantly better than that of the I/R group .p > 0.05). Compared with the sham group, the degree of myocardial fibrosis in the I/R group was more severe along with the extension of reperfusion time, especially from day 3 to day 30 (day 1: I/R vs. sham: (5.60 ± 2.30) (%) vs. (1.64 ± 0.41) (%), p < 0.05; day 30: I/R vs. sham: (29.81 ± 6.86) (%) vs. (1.74 ± 0.35) (%), p < 0.001). However, the degree of myocardial fibrosis in the I/R + antagomir-320 group was less severe than that in the I/R group at each time point   .vs. sham: (54.6 ± 7.3) (%) vs. (5.2 ± 2.1) (%), p < 0.05). The large amount of apoptosis may be due to the ischemia reperfusion injury, which was in line with the result of H&E staining. Compared with the I/R group, the apoptosis rate was significantly decreased in the I/R + antagomir-320 group   .p < 0.05), especially at 3 d after surgery (0.540 ± 0.134). Meanwhile, we observed that the expression of miR-320 dramatically decreased in the I/R + antagomir-320 group compared with the I/R group during day 3 to day 30 after surgery .qRT-PCR was applied to detect the expression of miR-320 in the three groups. Ischemic heart disease has been widely accepted as one of the most common and detrimental diseases, and surgical treatments for patients with ischemic heart disease do not mean complete myocardial ischemic reperfusion (I/R) and a normal internal environment . It has p/dtmax in the I/R group were apparently lower than those in the sham group, while the value of LVEDP was higher than that in the sham group. Furthermore, the values of LVEF, LVFS, LVSP and ±dp/dtmax in the I/R group gradually declined, but the LVEDP value gradually increased along with the extension of the time of reperfusion. Nevertheless, the values of LVEF, LVFS, LVSP and ±dp/dtmax in the I/R + antagomir-320 group were significantly higher when compared to the I/R group, indicating that the treatment of miR-320 may improve the heart function after myocardial I/R injury. Ischemic heart disease patients after therapy often suffer from myocardial reperfusion, which may cause acute myocardial damage and ventricular remodeling [2+ handling [2+ handling, and various studies have unveiled the essential role of miRs in modulating cardiac excitability and arrhythmogenesis [et al. observed that the values of LVEF and LVFS were decreased in the myocardial I/R injury tissues when compared with the sham controls, which was in line with our study results [In this study, our transesophageal echocardiography results have observed that the values of LVEF, LVFS, LVSP and ±dmodeling . Reoxygemodeling . Moreovemodeling ,37. In amodeling . Arrhythhandling . miRs fuogenesis ,41. Numeogenesis ,43. Fan  results . Further results . It has  results ; hence,  results . TherefoIn addition, our H&E staining results revealed that the I/R group exhibited extensive myocardial damage when compared with the sham group, while the degree of damage in the myocardial cells of the I/R + antagomir-320 group was significantly better than that of the I/R group. These results suggested that the decreased miR-320 expression level may decrease the degree of myocardial damage, reduce the risk of ventricular remodeling after surgery and, then, improve heart function. The reason why miR-320 was associated with the degree of myocardial damage may be that over-expression of miR-320 may downregulate the heat-shock protein 20 (Hsp20), which may protect the heart against myocardial I/R injury, while the decreased expression of miR-320 may reduce infarct size ,22. AnotOur TUNEL results also indicated that the apoptosis rate of myocardial cells in the I/R group was significantly higher than that in sham group, while the rate of myocardial cell apoptosis in the I/R + antagomir-320 group was apparently lower than that in the I/R group. These outcomes have suggesting that myocardial I/R may induce myocardial cell apoptosis and that treatment with miR-320 can reduce myocardial cell apoptosis. An increased expression level of miR-320 may lead to cell apoptosis or death and may increase infarct size after I/R injury, while the knockdown of miR-320 expression may have obviously protective effects on cells ,50. As pIn this study, the qRT-PCR results also indicated that the miR-320 expression levels in the I/R group were apparently up-regulated when compared with those in the sham group, suggesting that the miR-320 expression levels were closely associated with the myocardial I/R injury, and the increased miR-320 levels may have acted as a risk factor for myocardial I/R injury. miR-320 may play an important regulatory role in cell proliferation, cell differentiation and also cell apoptosis . FurtherHowever, several limitations in the present investigation still exist that merit our further exploration. Firstly, the trial size in the designed experimental rat model was relatively small, and the baseline characteristics shown in this paper were lacking regarding the adult male Wistar rats. Our study was focused on the animal model, which suggests that more experimental investigation among human populations is needed to contribute to the possibility of a target for the therapeutic intervention of myocardial I/R injury.All rats were purchased from the Shanghai experimental animal center of the Academy of Sciences of China, and the procedures of this animal experiment were approved by the Animal Research Ethics Committee of the Second Hospital of Jilin University . This study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983.The adult male Wistar rats weighing 240–280 g were anesthetized with 10% (0.4 mL/100 g) chloral hydrate by intraperitoneal injection before endotracheal intubation. Under aseptic conditions, the rats were placed in a supine position, and surface leads were placed subcutaneously. The electrocardiogram (ECG) monitoring was synchronously continued until the end of this experiment. After a left thoracotomy incision (1.5 cm from the third to fourth ribs), the left anterior descending coronary artery (LAD) was revealed by blunt dissection. A 6-0 nylon suture was placed around the left anterior descending coronary artery from the tip of the left auricle. Myocardial ischemia was performed by using a 6-0 nylon suture around LAD and tied for 60 min. Regional ischemia was confirmed by visual observation with the occluded distal myocardium of a pale color.n = 40); the rats underwent sham operation without coronary artery ligation; (2) the ischemia/reperfusion group , where the rats were treated with ischemia for 60 min; and (3) the I/R + antagomir-320 group (n = 40); 24 h before left coronary artery ligation, the rats were given an intramyocardial injection of antagomiR-320 (2 µg per day) at 1, 3, 5, 7 and 30 days after the operation.The 120 Wistar rats were randomly assigned into three groups: (1) the sham-operated group . When the picture on the screen was stabilized, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured from the parasternal long-axis view at the mid-papillary muscle level with the accompanying software.Echocardiogram indices of cardiac function and changes in hemodynamic parameters were recorded and analyzed using echocardiography at various time points  after surgery. Transthoracic echocardiography was performed by using a high-resolution p/dtmax) and maximum rate of left ventricular pressure decline (LVdp/dtmin) were recorded and measured by a BL-420F polygraph system .Hemodynamic measurement was conducted immediately after echocardiography. The rats were transferred to a clean experimental platform and connected with a small animal ventilator . The right carotid artery was separated into proximal, distal and mid-regions. The proximal portion of the carotid artery was occluded with a bulldog clamp, and the distal portion was ligated. After routine disinfection and draping, this artery was punctured, and then, a micro-pressure sensing catheter  was placed into the artery. Subsequently, the clamp on the carotid artery was loosened, and the catheter was placed into the left ventricle. Pressure recording equipment was turned on; left ventricular end-diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP), maximum rate of left ventricular pressure rise (LVdn = 3 sections per heart) under optical microscopy.After the hemodynamic examination, a thoracic operation was performed with ventilator support and ECG monitoring; the heart of the rat was excised, and the blood supply area of left anterior descending (LAD) artery was dissociated. Myocardial tissue of the infarct zone was collected quickly. At the end of the experimental period, the rats were euthanized by cervical dislocation. Tissues samples of rats were cut into two parts: half of the samples were snap-frozen in liquid nitrogen until use. The remaining samples were fixed in 4% neutral-buffered paraformaldehyde for 24 h. Sections 5 μm in thickness were paraffin embedded according to the standard procedure. Then, the samples were stained with the hematoxylin and eosin (H&E). The degree of heart damage and photographs were obtained from each heart section . In each section, digital photographs were taken at five randomly selected fields. The myocardial fibrosis area percentage was calculated by the ratio of the stained area to the total area. The calculation of the collagen volume fraction is as follows: collagen volume fraction  =  × 100%.in situ cell death detection kit  in accordance with the instructions of the manufacturer. Briefly, the enzyme, TdT, was applied to incorporate digoxigenin-conjugated dUTP into the ends of DNA fragments. The signal of TUNEL was measured by an anti-fluorescein antibody conjugated with alkaline phosphatase. Graded alcohols were used to dehydrate the slides, which were then coverslipped with hematoxylin for counterstaining. For each slide, color video images of 280–360 μm fields were captured and digitized by use of a ×25 objective with a Sony DXC-760MD video camera, a RasterOps 24 XLTV video card and Media Grabber software on a Macintosh computer. The cells with clear nuclear labeling were defined as TUNEL-positive cells. The apoptotic cells were calculated as the percentage of TUNEL-positive cells using the following formula: number of TUNEL-positive cell nuclei/ × 100%.Apoptosis of the heart sections was performed by terminal dUTP nick end-labeling (TUNEL) staining with an ® mini Kit RNA , in accordance with the manufacturer’s protocol. The cDNA was synthesized by using the TaqMan®Reverse Transcriptase Kit  with 3 μL of total RNA from each sample. The quantitative real-time RT-PCR was performed using the Maxima®SYBR Green/ROX qPCR Master Mix (2×)  in an ABI 7500 Real Time PCR System. The primers used for miR-320 in the present study were as follows: forward primer: 5'-AAAAGCTGGGTTGAGAGGG-3'; RTprimer: 5'-GTCGTATCCAGTGCGTGTCGTGG-AGTCGGCAATTGCACTGGATACGACTTCGCCCT-3'; reverse primer: 5'-TGCGTGTCGTGGAGTC-3'. All reactions were incubated at 95 °C for 10 min, followed by 40 cycles for 15 s at 95 °C and at 60 °C for 1 min. The U6 served as an internal control. Data for miR-320 copies were determined as the relative quantification, which was measured by using the 2Ct−ΔΔ method.We isolated the total RNA by using miRNeasy2 test was used to compare percentages. The Student’s t-test and one-way analysis of variance (ANOVA) were used for normal distribution variables. Non-normally distributed variables were calculated by the Mann–Whitney’s U-test. The Fisher exact test was used to compare nominal variables between the two groups. A probability value of less than 0.05 was considered as statistically significant.All data analyses were performed using SPSS 18.0 software . Data were expressed as the mean ± standard deviation (mean ± SD), frequencies or the median with interquartile ranges (IQR). The χTo summarize, our study results support the view that the expression of miR-320 might be up-regulated in the rat model of myocardial I/R injury, and miR-320 might contribute to the prevention of left ventricular remodeling after myocardial I/R injury. Consequently, decreased miR-320 expression may, in turn, be considered as a possible predictive factor in reducing the risk of left ventricular remodeling risk after myocardial I/R injury and improving cardiac function.


Based on the beta diversity of the operational taxonomic units (OTUs) in the inflammation and non-inflammation samples, the microbial variation in the oral cavity differed significantly between the two groups before and after the operation (P < 0.05). Analysis of the relative abundances of pre-operative OTUs revealed 26 OTUs with a relative abundance higher than 0.01%, reflecting a significant difference of the relative abundance between groups (P < 0.05). According to a principal component analysis of the pre-operative samples, the inflammation-related OTUs included Tannerella sp., Porphyromonas sp., Gemella sp., Moraxella sp., Prevotella nigrescens, and Prevotella intermedia, most of which were enriched in the inflammation group and showed a significant positive correlation. A cross-validated random forest model based on the 26 different OTUs before the operation was able to fit the post-operative status of grafted sites and yielded a good classification result. The sensitivity and specificity of this classified model were 76.9% and 86.7%, respectively. These findings show that the oral microbiota profile before alveolar bone grafting may be related to the risk of post-operative inflammation at grafted sites.In this study, we sought to investigate the oral microbiota structure of children with cleft lip and palate (CLP) and explore the pre-operative oral bacterial composition related to the prognosis of alveolar bone grafting. In total, 28 patients  with CLP who were scheduled to undergo alveolar bone grafting for the first time were recruited. According to the clinical examination of operative sites at the third month after the operation, the individuals were divided into a non-inflammation group (n = 15) and an inflammation group (n = 13). In all, 56 unstimulated saliva samples were collected before and after the operation. The v3-v4 hypervariable regions of the  Alveolar cleft, a symptom often occurring with cleft lip and/or palate (CL/P) with a frequency of 1.0–2.21 per 1000 live births . SecondaThe outcome of alveolar bone grafting is influenced by many factors, such as the operation timing, grafted bone sources, oral health status, and post-operative infection or inflammation ,7. AccorIntraoral surgery, including alveolar bone grafting, should be considered a ‘clean-contaminated operative procedure’, because intraoral surgical access inevitably suffers contamination of the surgical wound with the facultative pathogenic mixed flora of the oral cavity . The bac16S rRNA gene sequencing can detect the broad spectrum of both culturable and non-culturable microbiota in the oral cavity, providing insight into the diversity and community structure of the oral microbiome . Cl. Cl26].  < 0.05) .http://www.cs.waikato.ac.nz/ml/weka/index.html) using the different OTU abundance profiles with a relative abundance higher than 0.01% to assign saliva subjects to two groups (non-inflammation and inflammation).We performed a supervised classification using a cross-validated random forest model, based on the pre-operative OTUs differing between non-inflammation and inflammation samples –29. The The demographic and clinical parameters of the study subjects were shown in Firmicutes , Proteobacteria (31.2% in the inflammation group and 32.9% in the non-inflammation group), Bacteroidetes (17.8% in the inflammation group and 16.1% in the non-inflammation group), Actinobacteria (7.4% in the inflammation group and 7.4% in the non-inflammation group), and Fusobacteria (3.6% in the inflammation group and 2.5% in the non-inflammation group). These five predominant phyla constituted 98.1% of the total microbiota in the inflammation group and 98.2% in the non-inflammation group , the inflammation samples appeared to cluster together in the direction opposite to that of the non-inflammation samples . The infTannerella sp., Porphyromonas sp., Gemella sp., Moraxella sp., and P. nigrescens. The OTUs enriched in the non-inflammation samples were negatively correlated with a number of inflammation-enriched OTUs Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 File(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.


It has been documented that cytokines play important roles in the induction of normal functions of the placenta. It has been hypothesized that abnormal expression of the cytokines may be associated with unsuccessful pregnancy. The aim of this study was to compare the serum levels of interleukin-17A (IL-17A) and tumor growth factor (TGF-β) in pre-term, term neonates, and their corresponding mothers. This study was performed on 100 term and 60 pre-term neonates, and also on their corresponded mothers. Serum levels of IL-17A and TGF-β were examined by enzyme linked immunosorbent assay (ELISA). Our results revealed that the serum levels of IL-17A were significantly decreased in pre-term neonates in comparison to full-term neonates. However, the serum levels of IL-17A in the mothers either with pre-term or full-term neonates were not different. Also the serum levels of TGF-β were not changed in pre-term neonates and their mothers when compared with full-term neonates and their mothers, respectively.Based on these findings, it can be concluded that IL-17A may play crucial roles in induction of normal pregnancies and also probably participates in normal growth of fetus. Previous studies demonstrated that cytokines play important roles in regulating immune system functions including responses against infections and cancers, induction and suppression of autoimmunity and physiological functions of placenta -8. AdditInterleukin-17A (IL-17A) is one of the main cytokines which is produced by Th17 lymphocytes and stimulate several inflammatory conditions against microbes and during immune system-related diseases including chronic inflammation diseases and autoimmunity , 17. On Thus, the aim of this study was to examine the serum levels of IL-17A and TGF-β in pre-term neonates and their mothers in comparison with full-term neonates and their mothers.In this cross sectional study, cord blood samples of 60 pre-term and 100 term neonates were obtained. Plasma samples were also collected from the peripheral blood of their corresponding mothers at Rafsanjan Maternity Hospital of Nick-Nafs. All pregnant women with pre-term delivery and premature rupture of membrane, from February to September 2014, was introduced to the study as the case group and control group were selected randomly from the same age, gravida and parity women with term delivery . A gynecPre-term and term deliveries were determined based on the time of labor before or after 37 weeks respectively, based on the first day of last menstruation. The participants (control and case group pregnant mothers) were matched for age and the number of deliveries and those with bias factors including, pre-eclampsia, infection, smoking, diabetes, cesarean delivery, irregular menstruation, allergies, blood pressure and vaginal bleeding were eliminated from the study. All of the participants completed an informed consent form prior to blood sampling. The protocols for this study were approved by the Ethics Committee of Rafsanjan University of Medical Sciences.Detection of the serum cytokine levelsThe serum levels of IL-17A and TGF-β were examined using enzyme linked immunosorbent assay (ELISA)  technique immediately after blood collection according to the manufacturer’s instruction. The kit’s sensitivity was 2 pg/ml and the coefficient variation (CV) for inter- or intra-assay was identified to confirm the assessment reliability.Statistical analysisThe differences in variables were analyzed using independent sample t-tests  and Mann-Whitney U test  under SPSS software  and p˂0.05 were considered significant.Results of our study revealed that the mean IL-17A serum levels were 101.90±5.27, 79.25±2.64, 96.03±5.85 and 88.29±4.66 pg/ml in term and pre-term newborns and their corresponding mothers, respectively . StatistThe results also showed that the serum levels of TGF-β in term, and pre-term newborns and their corresponding; mothers were 70.22±10.99, 48.09±4.44, 58.34±9.09 and 48.00±8.54 pg/ml, respectively. Statistical analysis revealed that contrary to their mother (p=0.41), the differences between neonates (p=0.04) were significant . Our resThe results also revealed that neonate's weight, gestational age, and Apgar score of the preterm neonates are significantly different from normal term neonates (p<0.001) , while, It has been documented that cytokines play key roles in physiological and pathological conditions of pregnancy . IL-17A et al reported that serum levels of IL-17A in mothers with pre-term neonates were significantly decreased compared to mothers with full-term delivery (Therefore, according to our findings it appears that these cytokine may play important roles in induction of normal pregnancy and their down-regulation is associated with pre-term delivery. In agreement with our results, Hee delivery . It has delivery . It seemdelivery .et al reported that NKT cells also can induce pregnancy loss via up-regulation of IL-17A (Impaired function of NKT cells can result in unregulated expression of cytokines which may lead to pre-term delivery. Based on our results, it appears that these cells are unable to produce adequate rate of IL-17A and TGF-β which may be a reason for pre-term delivery. Additionally, Li f IL-17A . Severalf IL-17A , 26-28. f IL-17A .et al also reported that mother’s serum levels of TGF-β were not associated with spontaneous term or pre-term labor (It appears that two sources, NKT cells and T regulatory lymphocytes, play crucial roles in production of TGF-β to regulate immune responses against fetus antigens and also prepare appropriate conditions for fetal growth. Thus, based on our results, it may be concluded that down-regulation of TGF-β in fetus may lead to pre-term delivery. In contract with neonates, according to our findings, it appears that mother’s serum levels of IL-17A and TGF-β are not able to change the time of delivery. Tutdibi rm labor . Additiorm labor .The current study also confirms the inflammatory conditions in pre-term delivery which is associated with down-regulation of TGF-β, as an anti-inflammatory cytokine. Therefore, based on the results presented here and our results it seems that IL-17A and TGF-β can participate in outcome of pregnancy and more studies on the main mechanisms played by the cytokines can improve our knowledge regarding the roles of cytokines in pregnancy. Based on our results the serum levels of IL-17A and TGF-β were not differing between mothers. It appears that low sample size in our study is the main limitation to achieve a significant result. Additionally, based on the fact that cytokines play their function in a network format and according to our limitation in the project costs, hence, it appears that more cytokines should be evaluated to achieve a good conclusion. Therefore, more sample size and also evaluation of other cytokines which work in network format with IL-17A and TGF-β, such as IL-23, can improve our knowledge regarding the roles of cytokines in the preterm delivery. Additionally, our results demonstrated that the preterm neonate's weight, gestational age, and Apgar score were differ from normal term neonates  which is


Strong evidence shows that an increase in cardiorespiratory fitness (CRF) and physical activity (PA) reduces cardiovascular disease risk.To test whether a scientifically endorsed program to increase CRF and PA, implemented on an easy-to-use, always-accessible mobile app would be effective in improving CRF.Of 63 healthy volunteers participating, 18 tested the user interface of the Cardio-Fitness App (CF-App); and 45 underwent a 2-week intervention period, of whom 33 eventually concluded it. These were assigned into three groups. The Step-based App (Step-App) group (n=8), followed 10,000 steps/day prescription, the CF-App group (n=13), and the Supervised Cardio-Fitness (Super-CF) group (n=12), both followed a heart rate (HR)-based program according to American College of Sports Medicine (ACSM) guidelines, but either implemented on the app, or at the gym, respectively. Participants were tested for CRF, PA, resting systolic and diastolic blood pressures , resting, exercise, and recovery HR.P<.001). SBP decreased in all groups . DBP decrease was higher in the Super-CF group (-3.5 mm Hg) than in the Step-App group . Posttest exercise HR decreased in all groups . Posttest recovery HR was lower in the Super-CF group (-10.1 bpm) than in the other two groups . The CF-App group, however, achieved these improvements with more training heart beats (P<.01).CRF increased in all groups (+4.9%; A 10,000 steps/day target-based app improved CRF similar to an ACSM guideline-based program whether it was implemented on a mobile app or in supervised gym sessions. Low cardiorespiratory fitness (CRF) and physical activity (PA) have been shown to be two key independent risk factors for cardiovascular disease (CVD) and all-cause mortality -4. TheseTen thousand steps a day is a well-accepted PA goal, which has shown benefits in improving people’s cardiovascular health . The susNext, to the issue of what is the most effective training program to reduce CVDs risk, there is the problem of getting people to adhere to such a program. Low adherence to PA and exercise programs has been a known issue for decades . Many peRecently, smart, wearable, and mobile technology has enabled a large number of health applications . As it wKnight et al  reviewedTherefore, the primary aim of this study was to test whether a scientifically endorsed program to increase CRF implemented in an easy to use, and always-accessible mobile app, would be effective in improving CRF. A secondary objective of this study was to investigate how a HR-based training would compare with a steps-based training in terms of CRF changes.In order to test our research hypotheses a three group, pre- and posttest, 2-week intervention study was designed. The study protocol was approved by the Internal Committee of Biomedical Experiments of Philips Research as well as the Departmental Ethics Committee of the Milan University according to the Declaration of Helsinki.2. After signing the informed consent participants were screened for cardiovascular risk using the American Heart Association/ACSM Health/Fitness Facility Preparticipation Screening Questionnaire  · %intensity desired]+HRrest (3)Target HR=[ and classified as sedentary would receive a training frequency of three times a week, and a training session duration of 30 minutes, making a weekly mBeats target equal to target HR × session duration × frequency per week of 10,800 mBeats. The week was divided into training days and resting days. In this example, a training day would have a daily target of 3600 mBeats, while a resting day would have a target of 0 mBeats. The daily targets are not fixed, but depend on the remaining mBeats for the week. In other words, if the weekly target mBeats was 10,800 and on the first training day 7000 mBeats were already achieved, the daily target on the second training day would have been 1900 mBeats (the remaining 3800 mBeats divided by 2 remaining training days of the week).The participants in the Super-CF group were instructed to visit the gym three to four times per week according to their training program consistent with the ACSM guidelines described above. During the training session at the fitness center participants were supervised and motivated by an experienced personal trainer. They were also asked to wear the HR monitors and step counters for the whole day during the entire intervention period without a specific goal.A mobile app was specifically designed for the purpose of this study. Main features included in this first prototype of the CF-App were a HR feedback element, daily and weekly mBeats targets, an activity planner based on the theory of implementation intentions , and proOur App included a planning option, in which the participants could decide how to distribute their training days according to their own private schedule . This plUser interaction experience was tested during a 3-week pilot test by 18 healthy adults . The test started with a baseline week followed by two intervention weeks. During the baseline week, participants were instructed to wear the HR monitor and keep the app running, in order to collect physical activity data. All functionalities of the app were disabled and participants were asked to be as physically active as usual. After the baseline week, the 2-week intervention period started in which participants were coached to achieve their daily and weekly mBeats targets. At the end of the study, a one-on-one, semistructured interview was conducted to discuss usability and user experiences in depth. In addition, usability was measured with the Computer System Usability Questionnaire (CSUQ) . This qu2 max.The Statistical analysis was performed using the Statistical Package for Social Sciences (version 21) and the level of statistical significance was set at 0.05. Data were presented as means ± standard deviations unless otherwise indicated. Dependent variables were analyzed with a two-way, repeated, measures-mixed analysis of the variance (ANOVA), where the two factors were: time (pre- and post-intervention) and group . Because only the Step-App and the CF-App groups were randomized, a two-way, repeated measures ANOVA was performed also only on those two groups. If there had been violations of the sphericity, the corrections of Green-house Geisser if ɛ<0.75 and Huynh-Feldt if ɛ>0.75 were applied. The significant interactions were followed by post-hoc Tukey test. Pearson correlation coefficients were calculated between RDI and estimated VOOverall, participants were very positive about the potential of the CF-App and the concept of collecting heart beats to increase PA. Nonetheless, the pilot test revealed some points for improvement for the app. Most participants stated that their target zone was too narrow, and for some participants the zone was too high. Based on this feedback, the target HR zone was widened in the intervention study. Moreover, more features and functionality could be added to make the CF-App useful for people with various health goals. The current app is specifically designed to improve CRF, but over half of the participants stated they would want more information about the effects of all activities, including activities that are in a different HR zone. Furthermore, they wanted to be able to set their own exercise goals, to, for example, maintaining health or losing weight.Other than many other popular fitness apps, the CF-App stimulates users to achieve a weekly exercise target, rather than daily targets. The majority of participants  were in favor of a weekly target, as opposed to a daily target, because the amount of exercise they do is not the same for every day, but is fairly similar every week. Five of them stated that although they preferred a weekly target, they would be okay with having a daily target, as long as the daily target is integrated with what they have planned in their activity planner. Only three participants were in favor of the daily target because it would motivate them to get enough exercise on a daily basis. Overall usability from the CSUQ was toward the positive end of the scale. On a scale from 1 to 7, the participants rated usability 4.46 ± 1.46, quality of the interface 4.96 ± 1.26, system usefulness 4.72 ± 1.34, and information quality 4.21 ± 1.28.The ratings on the CSUQ questionnaire were in line with the qualitative findings obtained during the interviews. Ratings on the three variables were on the positive side of the scale, confirming the potential of the app. However, with an average rating of 4.5 on a 7-point scale, usability is not rated extremely high. This can mostly be accounted to some technical issues and the limited functionality of our first prototype. Moreover, participants identified missing elements that would make the app better, more interesting, and easier to interpret. Half of the participants explicitly stated they would add more parameters, such as speed and distance. This would make it easier for them to relate the mBeats to something they are already familiar with. Ten participants would like to add global positioning system tracking, or at least link the timestamps in the history view to a location, because it could give them insight in the effects of certain routes on their exercise performance.A majority of the participants mentioned that they would like advisory or motivational messages in the app. These could be tips on what kind of activities they could do to reach their target (8 participants), or information on how they are doing during exercising (6 participants). In addition, six participants mentioned they want to get messages about their achievements so far, like how many beats they still need to achieve, and another six participants wanted motivational messages such as ‘good job’ or ‘you have been idle for a while, isn’t it getting time to go for a jog?’Some participants did not fully understand the mBeats concept. They would need more education on advantages of the concept and measuring heart rate. Furthermore, they would need support in interpreting their data, to gain better understanding of their performance.Because people were uncertain about their current level of fitness and how to improve or maintain it, they would want the app to objectively assess their current fitness level and use this information to create a personalized training program, with feasible goals and reliable, and accurate HR zones.P=.63), nor main effect of time =0.189, P=.67) and of group =2.153, P=.13) in body weight. No time × group interaction was present for systolic =2.169, P=.08) and diastolic =0.426, P=.66) blood pressure. Although, a significant main effect of time was observed in both systolic blood pressure (SBP) =4.946, P=.03; Step-App: +1.19 mm Hg; CF-App: -3.23 mm Hg; Super-CF: -5.75 mm Hg) and diastolic blood pressure (DBP) =12.585, P<.001; Step-App: -2.12 mm Hg; CF-App: -4.31 mm Hg; Super-CF: -3.54); only the DBP showed a significant main effect of group =5.765, P<.01). Following-up these significant differences between groups it was observed that the DBP in the Step-App group was lower than in the Super-CF groups at baseline (P<.01) as well as at week 2 (P<.001) (P<.05), and a main effect of time for DBP =6.829, P<.05) were found.There were no significant time × group interactions =0.466, (P<.001) . When onP=.59). However, there were significant main effects of time =17.451, P<.001) and of group =5.380, P<.01). Although a significant main effect of time associated with positive deltas in all groups showed an overall improvement in CRF levels in all three groups , post-hoc analysis showed a significant difference between the Step-App group when compared with the Super-CF group (P<.001) =0.543, (P<.001)  A.P<.01). Follow-up analysis showed a significant difference at week 1 and 2 between CF-App and Super-CF group (P<.01 and P<.05) (P<.001). Post-hoc analysis underlined significant differences between the Step-App group and the Super-CF group during week 1 (P=.045) and week 2 (P=.03) (P<.001), showing a simple main effect of time in higher mBeats at week 1 and 2 in the CF-App (P<.001). There was a significant main effect of time in Ebbeling predicted CRF =8.814, P<.001). Steps counts showed two trends, one of a main effect of time toward an increase =2.994, P=.065) and the second one of a main effect of group =3.998, P=.060), but no interaction.A significant time × group interaction was found in week mean step counts =4.903, d P<.05)  B. In de (P=.03)  C. When P=.13). However, main effects of time =12.310, P<.001) and of group =11.132, P<.001) were observed. In particular the post-pre HR rest difference was significantly lower (P<.001) in the Super-CF group (-7.84 bpm); compared with Step-App group (-2.74 bpm) and the CF-App group (-3.33 bpm) (P=.36). However, there was a significant main effect of time =6.201, P=.02) but not between groups =1.683, P=.20). All three groups recorded lower HR peak due to the three different interventions  =2.169, .33 bpm)  D. The p.7 bpm )  D.P=.27; F=0.832, P=.49). HR measured after 1 minute revealed main effects within =9.318, P<.01) and between groups =16.798, P<.001). The Super-CF group had a significantly lower HR (-11.71 bpm) than both the Step-App  and the CF-App  groups. Also HR recovery measured after 3 minutes showed a main effect of group =7.989, P<.001). After 3 minutes, the HR in the Super-CF group (-10.17 bpm) was significantly lower than the one in the Step-App  and the CF-App group . Conversely, no within group differences were observed =1.634, P=.21).HR recovery after 1 and 3 minutes did not show significant time × group interactions =1.368, 2 max estimated using baseline values. A negative significant correlation was present between RDI and the estimated VO2 max . We then have used this as an additional marker of CRF. RDI did not show any time × group interaction =1.148, P=.25), but significant main effects of time =6.679, P=.02) and group =3.374, P=.03). The RDI was significantly lower in the Super-CF group than the Step-App (P<.05) and the CF-App (P<.01) groups  groups  D.P<.05) and the other for HR after 3 minutes =4.289, P<.05).When only Step-App and CF-App were compared, only two significant main effects of time were found. One for HR after 1 =4.619, Our research showed that a scientifically endorsed program to increase CRF, in line with ACSM's guidelines, implemented on smartphone in an easy to use and always-accessible app can improve fitness, and other health-related parameters. According to the treadmill walk test, CRF increased in all three groups not showing any interaction between the groups. Although, the weekly mean number of steps walked by the CF-App group did not increase drastically from baseline levels , and it may seem that participants in the CF-App group were more efficient than those in the other two groups in improving their CRF levels; yet no interaction and no main effect of group were found between the Step-App and the CF-App groups indicating that steps/day did not differ that much among the two App groups. The mBeats for the CF-App group did increase significantly from 43.5% of the mBeats target at baseline, 113.7% at week 1 and 105.5% at week 2, whereas mBeats in the Step-App group did not increase . Although the Super-CF group achieved a higher mBeats level , the participants of this group have done that by walking the highest number of steps per day. Most probably because these participants trained at the gym under the supervision of a personal trainer, three to four times a week. These results seem to suggest that intensity as well as volume training delivered by means of an easy to use mobile app accessible at any time, may be an efficient alternative to attending fitness classes. Although programs targeting steps can be a useful tool for sedentary people with a very low CRF level .Resting blood pressure results showed a small but significant decrease in SBP in the two intensity groups (CF-App and Super-CF), but not in the volume group (Step-App). DBP decreased in all three groups. Previous studies have found that both volume and intensity training are able to reduce blood pressure in hypertensive people . It is iResting as well as recovery HR did decrease, as expected , in all Our study had a number of limitations. Cardiorespiratory fitness was only indirectly estimated by using a treadmill submaximal test and a squat test ,31. The Our CF-App was built taking into consideration the user experience feedback. As shown in the Methods section an additional qualitative study was conducted on 18 people to improve the look and feel and the experience flow of our software app. Qualitative reports confirmed the high relevance and acceptance of our app. However, it could be improved by providing educational, interpretational, and motivational messages. Ultimately, it is important for people who have a low PA level and a low CRF to start doing something to improve their lifestyle behavior. Adhering one to one to guidelines and recommendations can be overwhelming for most people . By implAnother important aspect of our research was to encourage people to take any occasion throughout the day to engage in moderate to vigorous PA. For the less fit people this meant taking the stairs more often, cycling to work at a faster pace, or brisk walking during the lunch break instead of having a stroll. By no doubts motivation is key, and people with low readiness to change and self-efficacy will still struggle to adopt an active lifestyle. In the current study, given 100 as the maximal motivation to exercise, we had moderate levels in self-efficacy in all three groups, averaging approximately 54.3 ± 9.1. Yet all groups showed good short-term adherence throughout the two intervention weeks, still longer-term adherence, which is the hardest to achieve, remains to be investigated.For logistics reasons, participants were not randomly assigned to the Super-CF group. This has resulted in a baseline difference in CRF. However, this group was mainly used as a quality check of the intervention, to control for the main effect of time. This study, as mentioned above, was also kept rather short, only 2 weeks of intervention. We have shown in the past that sedentary people can improve their CRF when undergoing a vigorous but short training program . The choIn conclusion, a 10,000 steps/day target-based app improved CRF similar to an ACSM guidelines-based program whether it was implemented on a mobile app or in supervised gym sessions. Moreover, HR-based training improved CRF in equal measure as a steps-based training, but with a higher number of heart beats in a training zone for a similar number of steps/day than a Step-based training.


Force-displacement measurements are taken at different rates with an atomic force microscope to assess the correlation between cell health and cell viscoelasticity in THP-1 cells that have been treated with a novel drug carrier. A variable indentation-rate viscoelastic analysis, VIVA, is employed to identify the relaxation time of the cells that are known to exhibit a frequency dependent stiffness. The VIVA agrees with a fluorescent viability assay. This indicates that dextran-lysozyme drug carriers are biocompatible and deliver concentrated toxic material (rhodamine or silver nanoparticles) to the cytoplasm of THP-1 cells. By modelling the frequency dependence of the elastic modulus, the VIVA provides three metrics of cytoplasmic viscoelasticity: a low frequency modulus, a high frequency modulus and viscosity. The signature of cytotoxicity by rhodamine or silver exposure is a frequency independent twofold increase in the elastic modulus and cytoplasmic viscosity, while the cytoskeletal relaxation time remains unchanged. This is consistent with the known toxic mechanism of silver nanoparticles, where metabolic stress causes an increase in the rigidity of the cytoplasm. A variable indentation-rate viscoelastic analysis is presented as a straightforward method to promote the self-consistent comparison between cells. This is paramount to the development of early diagnosis and treatment of disease. The viscoelasticity of a cell reflects its function in an organism. Migratory cells, such as macrophages and neutrophils, are compliant. In contrast, osteoblasts, which generate bone, are stiff.  Interpla2614The complex elastic modulus of bulk soft materials, such as gels and tissue, is measured at a macroscopic level using rheometry. , 10 To m1414Changes in keratin  and otheTo maximize efficiency, cells compartmentalize their processes. Therefore, viscoelasticity is spatially heterogeneous across cells. For example, the leading edge of a migrating cell exhibits treadmilling of densely branched f-actin, while the f-actin concentration in the cytoplasm decreases.  Nuclear Herein, a scanning probe technique, which is based on an atomic force microscopy, is extended to accurately determine cell viscoelastic parameters with sub-micron spatial resolution. A variable indentation-rate viscoelastic analysis, VIVA, has advantages over force displacement (creep) measurements. This is because indentation curves are used to obtain spatially resolvable frequency sweeps. VIVA is used to detect viscoelastic changes in the cytoplasm of THP-1 cells induced by the toxicity of the silver nanoparticle, Ag NP. Dextran-lysozyme carriers (Dex-Gels) are used to deliver Ag NPs or rhodamine B to the THP-1 cells. In the fluorescent viability assay, rhodamine B and AgNP loaded Dex-Gels show some THP-1 cell toxicity. This correlates with an increase in cell stiffness and viscosity. However, the VIVA shows that there are no changes in the relaxation time of the cells. Furthermore, the ingestion of drug-free Dex-Gels, which show no toxicity in the fluorescent viability assay, does not change the viscoelasticity of the THP-1 cell cytoplasm. This rules out the possibility that the change in viscoelasticity observed with VIVA is simply due to the ingestion of Dex-Gel, and is actually correlative with cytotoxicity. These findings are significant to correlate changes in viscoelasticity with cytotoxicity at the single cellular level using AFM.Polyacrylamide gels (PAGs) with varying elastic moduli were synthesized to connect well-established bulk measurements of shear modulus to AFM nano-indentation measurements of elastic modulus. The PAGs were formulated using various combinations of acrylamide and bis-acrylamide concentrations (c.f. caption The AFM can be used to directly measure the elastic modulus (E) of PAGs by pressing on the surface of the gel with an AFM cantilever and measuring the cantilever deflection as a function of the indentation into the PAG. Deflection can be converted to force using the cantilever spring constant. It can fit to the Hertz equation for the appropriate indenter geometry, i.e., a sphere (discussion in supplement). In AFM nano-indentation, the strain-rate is the velocity at which the cantilever indents the PAG. The velocity of the cantilever, V, divided by the amount of indentation into the PAG, δ, determines the effective indentation rate in Hz. This can be plotted as a compressive analogue to the rheology data shown in The strain-rate dependence of a cell's elastic modulus has been previously established for a number of different cell types. In such studies, indentation at different strain-rates  and cree141 the high frequency modulus, E2, and the viscosity, η. These parameters can be extracted by fitting the frequency dependence of the cell modulus to the anticipated strain-rate response . This can be impregnated with toxic silver particles. Dex-Gels consist of a lysozyme inner core cross-linked with a dextran coating to form a 150 nm diameter nanoparticle that is digestible, capable of delivering drugs to cells and suitable for grafting specific proteins for targeted cell delivery.  Three vaTo be certain that self-consistent indentation was performed on a cell-by-cell basis, an inverted optical microscope with resolving capacity, which is sufficient to identify the nucleus, cytosolic region and cell periphery, was integrated with the atomic force microscope. The optical micrographs in 2 > 0.7) with the SLSM (solid traces). The cytoplasmic region is softest at all of the strain-rates and is comparable to the stiffness in the nuclear region. This is likely to be because the indentation probes a combination of stiffness of the nucleus and cytosol in this region. The cytosolic region represents the thicker region of the cell, up to 10 μm, which is optimal for nano-indentation (δmax = 0.5 μm ≪ 10 μm). The periphery is much thinner at circa 1 μm and may appear stiffer due to substrate effects (δmax = 0.5 μm ∼ 1 μm). The periphery is the stiffest region by twofold. All of the further modulus measurements were taken from the cytoplasmic region because this is where the Dex-Gels localize. This area is sufficiently thick for indentation and is wide enough to allow for measurements in multiple areas of the same cell. The nucleus is contained in this cytosolic region and was deliberately avoided to prevent overestimation of the modulus.Cell viability studies determine the health of the THP-1 cell populations after 24 h of treatment with the Dex-Gel . Calceinmax) remains 0.5 μm to avoid any indentation depth dependence of the modulus.Indentation of the THP-1 cells is performed across the relevant frequency range to determine the THP-1 cell viscoelasticity. The minimum and maximum indentation rates are chosen by determining where the modulus becomes independent of the strain-rate. For the THP-1 cells at velocities less than 0.2 μm/s and greater than 10 μm/s, the elastic modulus does not change significantly with the strain-rate. Viscoelastic materials, such as cells, display a maximum in hysteresis at a frequency that corresponds to the relaxation time. Thus, intermediate frequencies are selected by observing where the hysteresis between the indentation and retraction curves on the Dex-Gel treated THP-1 cells is maximized. Using this guideline, two intermediate velocities were selected: 0.5 μm/s and 1.0 μm/ s. Some hysteresis is also observed at the maximum and minimum indentation rates (supplement figure S3). This could be due to the viscoelasticity at these rates or the presence of short-range adhesion between the indenter and the cell. The AFM trigger point is adjusted during the measurement so that the average indentation depth  and Ag Dex-Gel (689 Pa) treated cells are greater than the control THP-1 cells, p <.001. Although the mean Ag Dex-Gel modulus is greater than the mean Rd Dex-Gel modulus, this difference is not statistically significant, p > 0.1.2 value is relatively low, 0.79. Similarly, for the Rd Dex-Gel treated distribution, the fit average is 551 Pa, a 10% underestimate, and the R2 is 0.8. While neither the Rd nor Ag Dex-Gel modulus distributions clearly evolve into bimodal distributions, stiff outliers and a reduced R2 suggest that anisotropic stiffening gives rise to stiffer subpopulations  correlates with the increased moduli of the Rd Dex-Gel and Ag Dex-Gel treated cells. This indicates that a stiffer subpopulation may arise, which is not apparent from a visual inspection of the data in ns c. f. . A corref = 20 Hz. At low frequencies, the modulus is also insensitive to frequency. At 0.4 Hz, the modulus value is approximately the zero-frequency elasticity. At intermediate frequencies (between 0.4 and 20 Hz), the transition between the low modulus and high modulus defines the viscosity of the cell. The strain-rate dependence of the elastic modulus is similar for all cell populations. A clear rate-independent increase in the average modulus is observed for the THP-1 cells treated with Rd Dex-Gel and Ag Dex-Gel. Over the range of tested strain-rates, the rate dependence of the THP-1 cells is consistent with the SLSM of viscoelasticity. Creep tests have shown that this is a good model for the stressrelaxation of multiple cell types. [In l types. –171, E2, and viscosity compared to the untreated and Dex-Gel treated THP-1 cells. The increase in E1 indicates stiffening, which is associated with immobile structures in the cytoplasm or the membrane stiffening. Meanwhile, the increase in E2 is indicative of an increase in the cell stiffness. This is associated with macromolecules that are mobile or bonds that release on the timescale longer than the measured relaxation time. The increase in viscosity suggests a reduction in the mobility of the cytoplasmic structures. The relaxation time appears to be conserved in the cell groups that are independent of treatment. This suggests that the mechanism that causes the cells to become stiffer also makes them proportionally more viscous. The scaling of τ may be attributed to the higher cross-linking density of cytoskeletal filaments. This increases the elastic stiffness and restricts the mobility of small molecules in the cytoplasm to flow away from stress . Furthermore, the E1, E2, and η values are in good agreement with results from AFM based creep tests. [p tests. –171, E2, η) directly to physiological processes or cellular components. E1 physically represents an entropic spring, which retains its elasticity (does not relax) independent of the timescale. Such stiffness may be attributable to less motile cell contents like the cell membrane, intermediate filament networks and cross-linked filamentous protein networks, which reversibly compress but return the cell to its original shape. The network of filamentous proteins, which is held within the cell membrane, exerts an entropic resistance to deformation. This depends on the length and concentration of the filamentous proteins in the cytoskeleton and could provide the anatomical basis for E1. Therefore, a non-zero E1 is more sensible than a zero elastic modulus at zero-strain-rate, which would describe a purely viscous cell. Creep tests have also shown the presence of a significant non-zero E1. [In addition to being spatially heterogeneous, the complexity of the cytoplasm makes it difficult to unequivocally relate the three parameters  and Sigma-Aldrich , respectively. Lysozyme and silver nitrate (AgNO3) were purchased from Sigma-Aldrich. No. 1 glass coverslips (25 × 40 mm2) and phosphate buffered saline (without Ca2+ and Mg2+) were purchased from Fisher Scientific .Analytical grade reagents were used, as received. Dextran with a MThe dextran-lysozyme conjugate hydrogels, Dex-Gels, are synthesized through a two-step heating process, as previously described. , 33 FirsTo prepare fluorescently labelled Dex-Gels, rhodamine B isothiocycyanate-and lysosome are mixed in buffer at a 1:1 ratio. The gelation protocol, which is described above, is then followed, yielding similar core-shell Dex-Gels with a rhodamine-B labelled shell.3 as a precursor, a solution of AgNO3 and Dex-Gel is autoclaved to reduce the Ag ions and nucleate AgNPs within the gel. Any outstanding AgNPs are separated from the Dex-Gel solution through a dialysis in deionized water. The subsequent Ag Dex-Gels are roughly 160 nm in diameter and contain 20.4 wt% silver composed of ∼ 5 nm diameter AgNPs. [Silver nanoparticles, AgNPs, are grown in Dex-Gels through an additional step following the above procedure to synthesize Dex-Gels. Using 25 mM AgNOr AgNPs. Polyacrylamide gels, PAGs, with defined elastic moduli are formulated using various mixtures of the monomer, acrylamide and the cross-linker, bis-acrylamide, in accordance with two previously set forth protocols. , 35 The N,N,N‘,N’-tetramethylethylenediamine, TEMED, is added to initiate free radial polymerization, while ammonium persulfate, APS, is added to catalyze the polymerization process. A concentration of 0.1 wt% TEMED and 0.1 wt% of APS are used for all solutions. Once the TEMED and APS are thoroughly mixed, the solution is drop-casted onto a chloro-silanated slide. An amino-silanated cover slip is dropped onto the chloro-silanated slide, creating a “sandwich” effect with the gelling solution in the middle. When polymerization is complete, the chloro-silanated slide can be peeled off, leaving a less than 500 μm thick PAG covalently grafted to the coverslip. The amount of solution drop-casted will affect the thickness of the gel but not the modulus.Piranha cleaned cover slips are coated with 3-Aminopro-pyltriethoxysilane, APTES, via physical vapour deposition, PVD. The cover slips are then placed in a 0.5% glutaraldehyde solution in PBS for at least 30 minutes. Excess APTES or glutaraldehyde is rinsed off thoroughly with PBS after their respective grafting. Piranha cleaned slides are chloro-silanated through PVD by dichlorodimethylsilane, DCDMS. Solutions containing different percentages of acrylamide and bis-acrylamide in PBS are used to develop PAGs with elastic moduli ratings spanning from 100 Pa to 50 kPa. PAGs prepared for rheology were drop cast ∼5 mm thick by pouring 1500 μL of polymerizing solution into a 25 mm diameter chloro-silanated glass boat. The gels were then removed from the boat and placed directly onto a RFS3 rheometer with PP25 chuck applying 200 g to the gel. Rheology was performed at 5% strain and 24.1 C between 0.01 and 500 Hz in triplicate. Frequency sweeps at 1% or 3% strain were found to return the same moduli as 5%.2) were plated onto glass coverslips (Ted Pella Inc.) in 35 mm polystyrene petri dishes . The THP-1 cells were stimulated with the addition of 1 μg/mL of Phorbol 12-myristate 13-acetate, PMA, for 72 h.THP-1 cells stably transduced with GFP-actin were maintained in a suspension culture in RPMI media , supplemented with 10% FBS , 1% penicillin streptomycin , and 0.05 mM 2-mercaptoethanol. ,36 For a2 in 35 mm polystyrene petri dishes . Prior to the experiments, the cells were allowed to grow for 48 h. The AFM analysis was carried out at room temperature (22°C) in HBSS, pH 7.4 with 1.3 mmol/l CaCl2, 0.9 mmol/l MgCl2, 2 mmol/l GlutaMax, 10 g/l heparin, 5.6 mmol/l glucose, and 1% FBS) analogous to C. M. Coll-Ferrer et al. [Human vascular endothelial cells were cultured in Vascu-Life VEGF Cell Culture Media  and checked for mycoplasma contamination using MycoAlert Kit . Adult human dermal fibroblasts  were cultured in Fibrolife cell culture media , as previously dsescribed.  The cellr et al. Following stimulation, the THP-1 cells were incubated with the Dex-Gels (20 μg/mL) for 24 h. As a control, the cells were incubated with media. After 24h incubation, the cells were washed with HBSS to remove the Dex-Gel from the solution. The viability of the untreated THP-1 cells, Dex-Gel and Ag Dex-Gel was determined by a fluorescence microscopy. The live cells were stained with calcein violet (750 nM) and the dead cells with ethidium homodimer (2 μM). The viability of the cells incubated with Rd Dex-Gels was determined by an optical microscopy following trypan blue staining (0.4 %). For each condition, five images were collected using a 10 × objective lens. The viable cells were counted using ImageJ . This experiment was performed in triplicate.An epifluorescence microscopy was performed using an Olympus IX70 microscope , outfitted with a Chroma Photofluor metal halide light source . Images were captured using a SensiCam QE camera  .IPLab software was used for image acquisition and to control the programmable filter wheels, shutters and focus . ImageJ was used for image analysis.2 micro-particle functionalized SiN cantilevers with nominal spring constant 0.08 N/m . Prior to each experiment, cantilever spring constants were manually calibrated in air using the MFP3D, using a clean glass substrate. The inverse optical lever sensitivity was determined in HBSS solution prior to each round of measurements. The elastic moduli were determined by fitting the indentation curve to the Hertz model, which was adjusted for a spherical indenter using the Asylum Research built-in analysis software. The deflection trigger for each indentation was adjusted prior to each round of measurements to obtain an indentation depth of 500 nm using several sacrificial cells.An optical microscopy and AFM were performed using an MFP3D AFM (Asylum Research), combined with an inverted Nikon TE-2000U microscope, equipped with a Nikon ×100, 1.49 NA objective. The Rd Dex-Gel fluorescence was captured via a 532 nm laser excitation (Crysta-Laser) and a CCD camera  with 200 ms exposure time, controlled through NIS Elements software package. All of the cells were observed with white light illumination from the MFP-3D during modulus characterization. A modulus characterization of the cells was performed using 2 μm diameter SiO1 and E1 + E2) increase proportionally. Using the SLSM, the VIVA enables three key parameters to be extracted from the indentation-rate dependence of the THP-1 cell elastic modulus: E1 is a measure of immobile elastic structures in the cell, such as the cell membrane rigidity and the f-actin concentration; E2 reflects the network cross-linking; and η reflects the mobility of this network related to cross-linking density and biofilament length. These experimental results should facilitate the development of universally comparable cell viscoelasticity models, which can be built up in complexity to further understand the molecular mechanisms that underlie cell biomechanics in health and disease.This study utilizes Dex-Gel drug carriers as biocompatible delivery agents of small molecule (rhodamine) and nanoparticle (Ag) toxins to stress THP-1 cells and observe the strain-rate dependent mechanical response by AFM indentation. A fluorescence microscopy shows that the Dex-Gels are incorporated into the cytoplasm of cells, where they are degraded. A viability assay indicates that the unloaded Dex-Gels are biocompatible , while the Ag Dex-Gel treated THP-1 cells become injured in association with the release of Ag in the cytoplasm . An AFM The authors have no conflict of interest to declare. No part of this study was performed on any human or animal subject.


Escherichia coli (n = 27), Klebsiella pneumoniae (n = 68) and Enterobacter spp. (n = 10) were isolated from blood of septicaemic neonates followed by antibiotic susceptibility tests, determination of MIC values, phenotypic and genotypic detection of β-lactamases. Carbapenem was the most active antimicrobial tested after tigecycline. CTX-M type was the most prevalent ESBL throughout the period (82%). New Delhi Metallo-β-lactamase-1 (NDM-1), which is a recent addition to the carbapenemase list, was the only carbapenemase identified in our setting. Fourteen percent of the isolates possessed blaNDM-1. Carbapenem non-susceptibility was first observed in 2007 and it was due to loss of Omp F/Ompk36 in combination with the presence of ESBLs/AmpCs. NDM-1 first emerged in E. coli during 2008; later in 2010, the resistance was detected in K. pneumoniae and E. cloacae isolates. NDM-1-producing isolates were resistant to other broad-spectrum antibiotics and possessed ESBLs, AmpCs, 16S-rRNA methylases, AAC(6′)-Ib-cr, bleomycin resistant gene and class 1 integron. Pulsed field gel electrophoresis of the NDM-1-producing isolates indicated that the isolates were clonally diverse. The study also showed that there was a significantly higher incidence of sepsis caused by NDM-1-harbouring isolates in the male sex, in neonates with low birth weight and neonates born at an extramural centre. However, sepsis with NDM-1-harbouring isolates did not result in a higher mortality rate. The study is the first to review the carbapenem resistance patterns in neonatal sepsis over an extended period of time. The study highlights the persistence of ESBLs (CTX-Ms) and the emergence of NDM-1 in Enterobacteriaceae in the unit.Treatment of neonatal sepsis has become a challenge with the emergence of carbapenemase-producing bacteria. This study documents the trend of carbapenem susceptibility in Enterobacteriaceae that caused septicaemia in neonates over a five year period (2007–2011) and the molecular characterisation of Enterobacteriaceae resistant to carbapenems and cephalosporins. Hundred and five Enterobacteriaceae including  Treatment of neonatal sepsis is a challenge. The treatment needs to be rapid, appropriate for the pathogen and safe for the neonate. The challenge seems to be increasing with each passing day due the escalating multidrug-resistant organisms Carbapenem resistance may occur due to expression of ESBL/AmpC-type enzymes combined with the decreased cellular penetration of carbapenems caused by loss of outer membrane protein. Isolates with this mechanism of resistance often express variable susceptibility to the different carbapenem agents. However, isolates with carbapenemase-mediated resistance are of special clinical concern because multi-institutional outbreaks have been reported worldwide Klebsiella pnemoniae carbapenemase , VIM  and OXA-48 (class D carbapenemase) Carbapenemases are enzymes that not only hydrolyse carbapenems but almost all hydrolysable β-lactams, and most are resistant against inhibition by the β-lactamase inhibitors blaNDM-1, can have up to 14 other antibiotic resistance determinants and can easily transfer this resistance to other bacteria The New Delhi Metallo-β-lactamase-1 (NDM-1) is the most recent addition to the list of carbapenemases. It is a zinc–requiring metallo–β–lactamase (MBL) that can hydrolyse all penicillins, cephalosporins, carbapenems and spares only the monobactam aztreonam Acinetobacter baumannii in the same unit This study was carried out in a neonatal intensive care unit (NICU) in which carbapenem resistance in Enterobacteriaceae was rare before 2008. Resistance to carbapenems was more a problem with lactose nonfermenting bacteria like The study protocol was carefully reviewed and approved by the Institutional Ethics Committee of the National Institute of Cholera and Enteric Diseases  (No. C-48/2010 T & E and NO. C-48/2011- T & E respectively). Individual informed consent was waived because this study used currently existing sample collected during the course of routine diagnosis of sepsis and did not pose any additional risks to the patients. The patient records/information was anonymized and de-identified prior to analysis.The study was conducted at a 20-bed level III unit of the IPGMER and SSKM Hospital, Kolkata, India between 2007 and 2011. The unit is the only Level III unit in the state. This unit has about 1000 admissions per year , including both intramural and extramural births.During 2007–2011, a total of 1985 blood specimens had been drawn from the admitted neonates suspected for sepsis on the basis of criteria set earlier by authors All Enterobacteriaceae isolated were identified by the ID 32 E kit . Antibiotic susceptibility profiles and minimum inhibitory concentrations (MIC) were evaluated along with phenotypic tests for the detection of β-lactamases and carbapenemases. Detailed molecular characterization and outer membrane permeability were carried out for the ertapenem-non-susceptible isolates. Molecular typing was performed only for carbapenemase-producing  isolates.Antimicrobial susceptibility testing was done by the Kirby-Bauer standard disk diffusion method The MIC values (mg/L) of cefotaxime, ertapenem, meropenem, amikacin, gentamicin and tigecycline were determined using Etest method  and were interpreted according to CLSI guidelines as modified in 2013. The clinical breakpoints for meropenem were as follows: susceptible (S) ≤1.0 mg/L, intermediate (I) 2.0–3.0 mg/L, and resistant (R) ≥4.0 mg/L. The same for ertapenem were as follows: S ≤0.5 mg/L, I: 1.0 mg/L, R ≥2 mg/L. MIC50 and MIC90 of meropenem were calculated as the MIC at which 50% and 90% of the isolates were inhibited.For all Enterobacteriaceae, the MIC value for ertapenem ≥0.5 mg/L was set as the screening breakpoint to detect carbapenemases The production of ESBLs, AmpCs, KPC and MBLs were evaluated using cephalosporin/clavulanic acid  combination disc, cefoxitin (30 µg)/boronic acid (300 µg)  combination disc blaVIM,IMP,SPM-1,GIM-1,SIM-1,KPC,SME,SPM,NDM,GES) blaSHV,TEM,OXA-1,CTX-M) blaMOX, CMY, DHA, ACC, MIR/ACT, FOX) http://www.ncbi.nlm.nih.gov/genbank).On the basis of results of the phenotypic tests, PCR was carried out for presence of carbapenemase genes  were done IntI1, IntI2 and IntI3 genes) were carried out for all 105 isolates For isolates resistant to either aminoglycoside, genotypic detection for 16S rRNA methylase-encoding genes (aac(6′)-Ib-cr and bleomycin resistant gene, bleMBL were also investigated in addition to other genes listed above bleMBL gene was further sequenced on both DNA strands to confirm its position with respect to blaNDM-1.In case of NDM-1 producing isolates only, association of plasmid-mediated quinolone resistance gene, E. coli (S205)  which retained both the porins (Omp C/F) has been used as a control for the western blots. Porins were detected using polyclonal anti-OmpC/F antibody as described earlier Whole-cell extracts of the ertapenem-non-susceptible Enterobacteriaceae isolates were separated on 11% SDS–polyacrylamide gels http://www.cdc.gov/pulsenet/protocols.htm) in a CHEF-DR III apparatus . XbaI macrorestriction patterns were compared and interpreted according to the criteria of Tenover et al.PFGE was carried out for all NDM-1-possessing isolates by following PulseNet standardized procedures , Klebsiella pneumoniae , Enterobacter cloacae  and one each of Enterobacter amnigenus and Enterobacter sakazakii (0.95%) were analyzed.During 2007–2011, 37% of the 285 culture positive isolates yielded Enterobacteriaceae. The 105 non-duplicate clinical isolates of Enterobaceriaceae including E. coli, with 100% susceptibility followed by carbapenems (74% for meropenem and 67% for ertapenem), over the five year period. All other broad-spectrum agents had susceptibility rates ranging between 22% and 74%   and the resistance was highest in 2011 (37.5% for meropenem and ertapenem both).Tigecycline was the most active antimicrobial tested against mikacin) . The resK. pneumoniae, tigecycline was again the most active antimicrobial with 96% susceptibility, closely followed by carbapenems (91% for meropenem and 87% for ertapenem). All other broad-spectrum agents had susceptibility rates ranging between 9% and 56% . (K. pneumoniae isolates did not appear until 2009 (30% for ertapenem) and the resistance to meropenem emerged in 2010. The resistance to carbapenems was highest in 2010 (33% for meropenem and ertapenem both). In contrast, in 2011, there was only 9% resistance towards carbapenems indicating decreasing resistance rates.In case of ikacin). . The resK. pneumoniae isolates are lower than E. coli isolates. But with respect to carbapenems, susceptibility rates for K. pneumoniae isolates are higher than E. coli isolates during the study period , the highest resistance was observed with cefotaxime (100%) followed by amikacin and gentamicin (87.5%), ertapenem (50%) and meropenem (37.5%). Tigecycline was the only agent for which 100% susceptibility was found. The resistance to ertapenem in E. cloacae isolates emerged in 2008 (n = 1) but resistance to meropenem was observed in late 2010 (n = 1); and was highest during 2011 (n = 2). Due to small number of Enterobacter isolates, percentage calculation as well as MIC50, MIC90 determination was not carried out. Enterobacter amnigenus and Enterobacter sakazakii were susceptible to all antimicrobial agents tested in this study.For E. coli and K. pneumoniae isolates for each year has been depicted in The MIC values inhibiting 50% (MIC50) and 90% (MIC90) of the organisms tested against cefotaxime, ertapenem, meropenem, amikacin, gentamicin and tigecycline are presented in E. coli isolates from 2007 to 2011. Since for E. coli, the sample number is small, particularly for the numbers in an individual year, the MIC50 and MIC90 obtained in this study should not be overstated as a few strains with high MICs may skew the result. For K. pneumoniae isolates, the range of MIC values and MIC90 values indicated an abrupt upward shift from 2009 to 2010, which did not persist in the following year (2011).The distribution of the MIC90 values indicated an upward shift of meropenem MICs among the E. coli and K. pneumoniae isolates remained within the susceptible range based on the CLSI guidelines as modified in 2013. In addition, a comparison of the two organisms revealed that the number of E. coli isolates non-susceptible to meropenem increased constantly throughout the study period while among K. pneumoniae, non-susceptible isolates were first detected in 2010 but the number of such isolates declined in 2011.MIC50 values for both As the MIC value for ertapenem ≥0.5 mg/L was selected as the screening breakpoint for carbapenemases, a comparison of the susceptibilities to other antibiotics  among ertapenem-susceptible and ertapenem-non-susceptible isolates  was carrE. coli, 4 K. pneumoniae, each of Enterobacter amnigenus and Enterobacter sakazakii) were susceptible to all generations of cephalosporins, monobactam and carbapenems as tested by the disc diffusion test. The remaining Enterobacteriaceae (n = 95) were further analysed for production of β-lactamases by phenotypic tests  produced MBLs. These fifteen isolates were also non-susceptible to meropenem (>1 mg/L). MBL-producing bacteria showed inconclusive phenotypic results for ESBLs and AmpCs. Therefore, the presence of ESBLs and AmpCs in these isolates was confirmed by PCR subsequently (Twenty-six isolates with elevated ertapenem MICs (≥0.5 mg/L) were considered for KPC and MBL analysis. None were positive for KPC production but fifteen isolates , two AmpC types (CMY and ACT) and only one carbapenemase type (NDM) . The mosSequencing for all the genes on both strands have been carried out in the ertapenem-non-susceptible isolates. NDM-1 was present in 14% (n = 15) of the isolates and was the only carbapenemase type identified. No other carbapenemases were detected in this study. Class 1 integron was observed in 69 isolates (66%).K. pneumoniae in 2 cases of neonatal septicaemia in 2010 E. coli had actually emerged in 2008 and much later (2010) in K. pneumoniae.We had earlier reported the presence of NDM-1 in K. pneumoniae, 2 E. coli and 2 E. cloacae) out of twenty-six ertapenem-non-susceptible isolates did not produce NDM-1 or any other carbapenemases. Microbiological and molecular characterization of these isolates has been documented in E. coli)/OmpK36 (in K. pneumoniae). A loss of porin in combination with ESBLs/AmpCs is the reason for carbepenem-resistance in these isolates; these mechanisms of resistance have been documented earlier by other authors also Eleven (7 armA or rmtB and plasmid mediated quinolone resistant gene aac(6′)-Ib-cr. This result indicates that the NDM-1 possessing isolates are associated with unrelated broad-spectrum resistance genes, suggesting that they have been selected by wide range antibiotics. Two novel β-lactamases were also identified in two isolates harbouring NDM-1, a new SHV-type, SHV-167 (GenBank accession no. AB733453) and an AmpC gene, ACT-16 (GenBank accession no. AB737978). The presence of these novel β-lactamases has been reported bleMBL immediately downstream of the blaNDM-1 gene. This association has been quite systematically identified throughout the world The molecular characterization of NDM-1-producing Enterobacteriacae (n = 15) is represented in The fact that class1 integrons were detected in nearly all isolates harbouring NDM-1 makes the situation even more worrisome. Class1 integrons are important players in driving the evolution of complex and laterally mobile multidrug-resistant units The distribution of different classes of resistant determinants among NDM-1-harbouring isolates and isolates not harbouring NDM-1 is described in The wide range of MIC values (1.5–≥32 mg/L) for meropenem among NDM-1 positive isolates prompted us to examine whether loss of porin was associated with such differences.K. pneumoniae (K1-K6), 3 E. cloacae (EC1-EC3) and 1 E. coli (E1) isolates (K. pneumoniae. All the porins were detected in 5 E. coli (E2-E6) isolates with MIC values 1.5 mg/L, 8 mg/L, 16 mg/L, 32 mg/L and ≥32 mg/L. Therefore, loss of porin seemed to be species specific and the differences in MIC values of the NDM-1 possessing isolates probably did not result due to absence of porins. Further work is in progress to understand the reason for such differences which can occur due to alterations in the expression of the enzymes or other changes in the outer membrane proteins.All 15 NDM-1-producing isolates retained normal levels of OmpA, a structural protein but OmpF/OmpK36 was not detected in 6 isolates . OmpF/OmHowever, it should be noted that all eleven ertapenem-non-susceptible isolates showed loss of porin which along with the ESBLs was the cause for carbapenem-nonsusceptibility.PFGE revealed that all NDM-1 carrying Enterobacteriaceae isolates were clonally diverse  and mostAn evaluation of the demographics and clinical data  revealedCefotaxime along with amikacin or gentamicin was used in the NICU as a pre-emptive antimicrobial therapy for clinically suspected cases of sepsis during 2007–2008. This was changed to piperacillin/tazobactam and amikacin due to the high prevalence of CTX-M gene in the unit. For serious cases of infection, meropenem, ofloxacin and colistin in different combinations were used particularly after the emergence of carbapenem resistance.The results of a comparison of neonates with presence of NDM-1-producing bacteria in their blood sample and those without NDM-1 are shown in In order to get an indication of the association between the presence of NDM-1-carrying Enterobacteriaceae in blood with mortality, a simple association analysis between the two was conducted. The results showed that neonates with NDM-1-harbouring Enterobacteriaceae in their blood actually had a mortality rate of 13.33% (2 out of 15 neonates). In comparison, the neonates without NDM-1-possessing Enterobacteriaceae in their blood had a mortality rate of 22.22% (20 out of 90 neonates). The difference in the mortality between septicaemic neonates with and without the presence of NDM-1-producing Enterobacteriaceae was not statistically significant [P-value 0.6595].As there are no previous studies that have analysed the association of clinical factors with sepsis due to NDM-1-carrying organisms in neonates, comparisons with earlier studies is not possible. Comparisons could only be made with studies where risk factors for neonatal sepsis have been investigated. One particular study has shown that male sex is associated with sepsis in neonates Sepsis caused by NDM-1-producing Enterobateriacae was not associated with mortality of the neonates in this study though one particular study in adults have shown that infections with carbapenem- resistant isolates had a higher mortality rate The emergence of carbapenem resistance particularly in Enterobacteriaceae is a considerable burden on the neonatal healthcare system in developing countries. This is the first analysis of the carbapenemases in a neonatal intensive care unit for an extended period of time. The study tries to capture the trend in resistance for a period before and after the emergence of NDM-1 in the unit. The study shows the persistence of the CTX-M-15 gene throughout the five year period. In fact, a prelude to carbapenem resistance, as observed in this study, has been the presence of ESBLs particularly CTX-Ms. Earlier studies from our laboratory and other studies from India have shown the extensive dissemination of this gene K. pneumoniae and E. coli displayed differences in susceptibility patterns. K. pneumoniae showed higher susceptibility to carbapenems but lower to cefotaxime, gentamicin and amikacin. Such differences in the species has also been noted in other studies E. coli isolates, particularly in individual years, these differences should not be overstated.Though present in the same setting, Though a number of variants of the NDM gene have been reported till date, no variants of this gene were identified in this study. All isolates possessed NDM-1. In addition throughout the five-year period no other MBL was detected in these isolates. The diversity of the isolates indicates probable horizontal transfer of the NDM-1 gene either through plasmids or by the transposons related acquisition. The capability of NDM-1 to associate with other resistance genes raises serious concerns. The other cause for concern is the difficulty in detection of ESBLs in the presence of NDM-1 which hydrolyse carbapenems and also cephalosporins.The high prevalence of ESBLs and the increasing presence of carbapenemases, both of which can be attributed to horizontal transfer, are indeed worrisome. Added to this is a vulnerable group of critical patients, the hectic environment of the ICU where lapses in infection control may happen and extremely capable pathogens. This is a fatal combination and necessary systemic steps need to be implemented soon.Figure S1Schematic representation of the molecular analysis of Enterobacteriaceae isolates enrolled in this study.(TIF)Click here for additional data file.Figure S2Graphical representation of percentage of Enterobacteriaceae isolates producing ESBLs, AmpC and NDM during the study period.(TIF)Click here for additional data file.Table S1Susceptibility patterns of the ertapenem susceptible and non-susceptible Enterobacteriaceae isolates for 4 broad spectrumantibiotics.(DOCX)Click here for additional data file.Table S2Microbiological and molecular characterization of 105 Enterobacteriaceae isolates included in this study.(DOCX)Click here for additional data file.


By Cox regression analysis the hazard ratio (95%CI) for disease-recurrence was 3.8  for HPV-mRNA-positive compared to HPV-mRNA-negative patients. After adjustment for tumor size as the most influential covariate the HR was still 2.8 . In patients with cervical cancer and tumor-free lymph nodes by conventional histopathology HPV-mRNA-positive SLN were of prognostic value independent of tumor size. Particularly, patients with tumors larger than 20mm diameter could possibly benefit from further risk stratification using HPV-mRNA as a molecular marker.Up to 15% of patients with cervical cancer and pN0-status develop recurrent-disease. This may be due to occult metastatic spread of tumor cells. We evaluated the use of human-papillomavirus-(HPV)-mRNA as a molecular marker for disseminated tumor cells to predict the risk of recurrence. For this prospective, multi-center prognostic study, 189 patients free of lymphnode metastases by conventional histopathology could be analyzed. All patients underwent complete lymphadenectomy. Of each sentinel node (SLN) a biopsy was taken for the detection of HPV-E6-E7-mRNA. Median follow-up time after surgery was 8.1 years. HPV-mRNA could be detected in SLN of 52 patients (27.5%). Recurrence was observed in 22 patients. Recurrence-free-survival was significantly longer for patients with HPV-negative SLN (log rank  In patients with cervical cancer, metastatic spread to lymph nodes is known to be the most important prognostic parameter which is crucial for selection of therapy. The risk of lymph node metastases in women with early stage cervical cancer (up to FIGO stage IB1) is approximately 15% and as a consequence the majority of patients do not benefit from lymphadenectomy . RadicalThe identification of ITC, in particular single tumor cells, requires a highly specific marker. In this context, the detection of cytokeratins by immunohistochemical staining or at the mRNA level is not ideal , 12. MorWe enrolled 189 out of 338 eligible patients Figure . The proClose to 60% of the primary tumors were HPV16 positive, 22% were HPV18 positive and the remaining tumors were either HPV35-, HPV45- or HPV73-positive. HPV-mRNA of the respective HPV type could be detected by RT-nested-PCR in SLN of 52 patients (27.5%). 119 patients (63%) had negative and 18 patients (9.5%) had probably negative findings . Five years after surgery 94.8% (95% CI 88.8 – 97.6%) of these patients survived without relapse compared to 80.2% (95% CI 66.2 – 88.8%) of patients with positive SLN  for HPV positive compared to patients with HPV-mRNA negative SNL. In single factor models tumor size was the most influential prognostic variable. Compared to patients with small tumors the risk of disease recurrence was 9-fold and 20-fold if tumor size was >20 to 40mm and >40mm, respectively. Grading and histology were not significantly associated with recurrence-free survival. The significant effect of HPV-mRNA in SLN was preserved in all two-factor Cox models. As expected from single factor models, the largest change in estimate was observed after inclusion of tumor size. HR for HPV positive compared to negative patients decreased to 2.8 . The independent prognostic values of tumor size and HPV-mRNA in SLN are shown in stratified Kaplan-Meier curves  and a tumor size adjusted HR of 2.5  was estimated  the crude and tumor size adjusted HR was 2.8  and 2.5 , respectively. The sensitivity analysis (n = 189) revealed a crude HR of 2.5  which decreased after tumor size adjustment to 2.1 .Post-hoc we analyzed the effect of HPV-mRNA in SLN on total mortality. In the primary analysis population . By Cox-regression analysis the hazard ratio  for disease recurrence was 3.8  for HPV-mRNA positive compared to negative patients. Most importantly, after adjustment for tumor size as the most influential covariate for recurrence, the hazard ratio was still 2.8 .This is the first multicenter prospective study which addresses the prognostic potential of HPV-mRNA in SLN of patients with pN0-status by systematic lymphadenectomy and conventional histopathology. We could show that recurrence-free survival was significantly longer for patients with HPV-mRNA negative SLN  in a background of 10otal RNA . In termotal RNA . As expep = 0.02) and marginally in grading (p = 0.045). However, both characteristics did not influence recurrence-free survival in our study. Additionally, recurrence-free survival of the excluded subjects was similar at the regular end of the study (log rank test p = 0.42). Second, the power of our statistical analysis was lower than assumed. Moreover, the initial follow-up period of 3 years for recurrent disease was too short and had to be prolonged. The final number of 22 patients with recurrence was still lower than expected. Therefore the number of simultaneous covariates in the Cox-regression model had to be restricted and may have resulted in incomplete control of confounding. Third, the HPV-mRNA status of SLN was unclear in eighteen patients. For these cases the SLN count by the surgeon did not match with the number counted by the pathologist. The SLN available for HPV-mRNA analyses however were negative. When we included the data of patients with equivocal results, the sensitivity analysis revealed a weaker but still significant association  we decided to prolong the follow-up period. From July to November 2013 data on vital and recurrence status could be updated for 130 patients out of 172 who had no recurrence during the regular study period. Now, the median follow-up time was 8.1 (range 0.5 to 12.5) years. Here, we present results of our final analysis. Although initiated much earlier this study fulfils the criteria recommended for tumor marker prognostic studies (REMARK) published in 2005 [This study was performed as the longitudinal, prognostic part of a, to date, largest prospective, multi-center diagnostic study which addressed the SLN-concept in cervical cancer . The stuInclusion criteria were all stages of invasive cervical cancer, intention of curative surgery and complete pelvic lymphadenectomy after removal of SLN (for details see ). FurtheThe primary endpoint was disease recurrence defined as local recurrence of primary disease, distant metastasis, death due to cervical cancer or death with unknown cause. In patients without an event observations were censored at date of last follow-up information that excluded disease recurrence. We primarily focused on the prognostic value of HPV-mRNA in pelvic SLN. HPV-mRNA-status was classified as positive, negative or probably negative. The last category was assigned to HPV-mRNA negative cases in which the SLN count by the surgeon did not match with the number counted by the pathologist. Tumor size , histology, grading and, age (post-hoc due to prolonged follow-up) were considered as additional prognostic variables.Patients underwent complete pelvic lymphadenectomy either by laparoscopy or by open approach (for details see ). The peHPV-genotyping was performed using genomic DNA extracted from frozen biopsy material obtained from primary tumors. Thirty-seven different genital HPV types, including all hrHPV types can be amplified by the GP5+ and GP6+bio assay .n=30VN-3′), 100nM of each dNTP, 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl2 10mM DTT, 20U RNasin und 200U SuperScriptII (GibcoBRL). To allow optimal annealing, primer and RNA were incubated at 70°C for 10 min and cooled down on ice. The remaining reagents were then added in form of a master mix and cDNA synthesis was done at 42°C for one hour. The reaction was stopped at 70°C for 15 minutes and stored at −80°C until PCR analyses.Approximately 30mg of tissue were homogenized (OMNI-TH homogenizer with dispersible tips) and total RNA was extracted thereof using the RNA Blood Mini Kit from Qiagen  according to the manufacturer's instructions. DNase treatment was included. RNA concentration and quality were determined by spectrophotometry (NanoDrop ND-1000) and gel electrophoresis. One microgram of total RNA was reverse transcribed in a 20μl reaction comprising 20pmol CDS-primer  PCR, 2μl of the first reaction (1:3 dilution in H2O) was used. The reaction conditions were the same as for the first PCR except for the use of inner primers. All reactions were run in a Mastercycler Gradient . PCR products were separated in 1.2% agarose gels for Southern blot analysis. Specificity was confirmed by hybridization using internal oligonucleotide probes (For the detection of hrHPV-E6-E7 specific cDNA a nested-PCR protocol was used. By this approach one tumor cell (HPV16 positive cell line SiHa) can be detected in a background of 10otein E7 . Outer ae probes .To control for successful reverse transcription 1μl of cDNA  was taken for the amplification of GAPDH. The primers for GAPDH were: GGTGAAGGTCGGAGTCAACG (forward) and CAAAGTTGTCATGGATGACC (reverse). Reaction conditions were as for hrHPV-E6-E7 amplification but not in the nested format. Successful amplification was assessed in agarose gels.n = 77). This assay was described in detail by Häfner and colleagues (16). Target gene expression measured in duplicate was normalized to the geometric mean of the expression of the two most stable housekeeping genes in lymph nodes .Beside RT-nested-PCR which provides a highly sensitive but only a qualitative readout, we also performed quantitative real-time analysis (qRT-PCR) for a subset of SLN (2 test, log rank test and Wald test as appropriate. For comparison of laboratory methods the kappa statistic was calculated. The two-sided level of significance was 0.05. All data were managed in an ACCESS database and were analyzed with statistical software SAS 9.3 and Stata 12.0.Sample size was calculated according to the rule of ten events per variable for proportional hazards regression analysis . Beside